PROJECT SUMMARY/ABSTRACT Antibodies constitute a powerful weapon to fight viral infections. They could neutralize the infectivity of virus particles as well as mediate killing of productively infected cells via mechanisms such as antibody-dependent cell-mediated cytotoxicity (ADCC). In the past five years, our group has utilized the advances made in the antibody engineering field, as well as the growing lists of broadly neutralization antibodies (bNAbs) identified by other investigators, to construct new bispecific antibodies that have exquisite antiviral breadth and potency for the purpose of HIV-1 prevention. Now, with this proposal, we wish to expand our antibody engineering effort to generate a collection of bispecific and trispecific antibodies that are optimized for killing of Env-expressing cells, and for important properties such as pharmacokinetics. Instead of screening for virus-neutralization activity as we have previously done, we will now engineer and screen a library of Env-targeting multi-specific antibodies for cell-binding and cell-killing activities in vitro. The best performing antibody constructs will then be evaluated systematically in vivo in a humanized mouse model for the effect on their cell-killing capacity, their ability to restrict or eliminate latent reservoir cells after activation, and their ability to prevent or limit the establishment of the HIV-1 latent reservoir. Along with our knowledge of HIV-1 bNAbs and antibody engineering, our deep understanding of viral dynamics and our prior experience studying the HIV-1 latent reservoir will be brought to bear on the design, conduct, and interpretation of experiments to evaluate and quantify the antiviral effects of our top antibody constructs in humanized mice. Our group has successfully engineered two bispecific antibodies with exquisite HIV-1-neutralizing activity, and we have since extended our know-how in antibody engineering to the construction of bispecific or trispecific antibodies that target Env for the purpose of facilitating the elimination of infected cells. In the end, we hope to offer to the field one or two multi-specific antibodies that could be applied toward the elimination of latent reservoir cells as one critical component of a multi-pronged approach to HIV-1 eradication.